Insulin is an anti-natriuretic hormone in the kidney. Insulin increases sodium reabsorption in the proximal tubule, thick ascending limb, and distal tubule. However, the molecular mechanisms underlying its actions are not well described. In addition, hyperinsulinemia, due to insulin resistance, is highly correlated with hypertension, which often precedes chronic renal disease. Nevertheless, because of the complexity of these pathological states, the direct effects of insulin, in the kidney through its own receptor, are difficult to parse out. Thus, these studies are aimed at developing two new animal models that will allow us to better address the direct role of insulin in the kidney and its role in NaCI balance, blood pressure, and the regulation of renal NaCI transport proteins. In order to achieve these aims our plan is to develop, through breeding, using the CRE/IoxP approach, two lines of transgenic mice in which the insulin receptor (IR) is knocked out of either the collecting duct principal cells or the thick ascending limb cells, respectively. These mice (and cell cultures prepared from their kidneys) will be evaluated for insulin-sensitive NaCI transport, blood pressure changes, and the regulation of target transport proteins. In this proposal, we test the overall hypothesis that mice that lack insulin receptors in either their collecting duct principal or thick ascending limb cells due to selective knock out of gene expression in these cells will have decreased NaCI reabsorption at these sites under basal or stimulated conditions. In specific aim 1, we plan to develop the collecting duct principal cell insulin receptor knock-out mouse (CD-IR KO) and evaluate NaCI transporting characteristics under basal conditions. In specific aim 2, we plan to develop the thick ascending limb cell insulin receptor knock-out mouse (TAL-IR KO) and, likewise, evaluate basal NaCI transport. Finally, in specific aim 3, we plan to evaluate blood pressure, NaCI balance, NaCI transport, and the regulation of renal NaCI transport proteins in the above mice under hyperinsulinemic conditions in vivo and ex vivo. We plan to achieve hyperinsulinemia in the mouse in two ways: 1) we will infuse insulin via the jugular vein; and 2) we will feed a diabetogenic diet that should induce insulin resistance and thus raise endogenous insulin levels. Overall, we believe these models will provide fresh molecular insight into a physiological phenomenon that has been known for decades, insulin induced hypertension, with clear clinical ramifications.